CN111936230A - Method for immobilizing ligand having amino group - Google Patents

Method for immobilizing ligand having amino group Download PDF

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Publication number
CN111936230A
CN111936230A CN201980007805.XA CN201980007805A CN111936230A CN 111936230 A CN111936230 A CN 111936230A CN 201980007805 A CN201980007805 A CN 201980007805A CN 111936230 A CN111936230 A CN 111936230A
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Prior art keywords
ligand
less
adsorbent
target compound
group
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铃木琢磨
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Kaneka Corp
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Kaneka Corp
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    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
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    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Abstract

The present invention provides an immobilization method that firmly immobilizes a ligand and is excellent in deactivation of residual formyl groups. The method of the present invention for immobilizing a ligand having an amino group and having a specific affinity for a target compound on an insoluble substrate containing a formyl group is characterized by comprising the steps of: a step of mixing the ligand and the insoluble substrate containing a formyl group to form an imine, and a step of reducing the imine by using 2 or more reducing agents.

Description

Method for immobilizing ligand having amino group
Technical Field
The present invention relates to a method for efficiently immobilizing a ligand having an amino group to an insoluble substrate having a formyl group.
Background
Biologically active substances such as peptides and enzyme substrates having specific affinity for a specific compound are immobilized on an insoluble substrate, and thus the substance that interacts with the immobilized biologically active substance can be recovered and detected, and therefore, the utility of the substance can be improved. For example, in affinity chromatography, only a target compound can be efficiently recovered from a mixed solution by immobilizing a bioactive substance that specifically binds to the target compound as a ligand to insoluble porous particles. Examples of industrial applications of affinity chromatography include: immunoglobulin separation using immobilized proteins, and antigen separation using immobilized antibodies.
As a method for immobilizing a ligand on an insoluble substrate, it is extremely important to perform immobilization by a strong covalent bond for industrial use in order to reduce leakage of the immobilized ligand. At the same time, the state of the immobilized ligand is also important, and the ligand is preferably immobilized in a state where the activity is maintained.
As a method for immobilizing a ligand to an insoluble substrate, for example, a method comprising: a formyl group is introduced into an insoluble substrate, and the formyl group is reacted with a ligand having an amino group to form an imine, and the imine is fixed by a reductive amination reaction in which the imino group is reduced to form a stable amine (patent document 1).
Further, a method is disclosed in which various specific reducing agents shown in patent document 2 are used in the same reaction, whereby the amount of leakage of the ligand can be significantly suppressed.
Documents of the prior art
Patent document
Patent document 1: japanese patent laid-open publication No. 2015-110224
Patent document 2: international publication WO2017/034024 pamphlet
Disclosure of Invention
Problems to be solved by the invention
However, the present inventors have found that the method of patent document 1 has room for improvement in the amount of ligand leakage, and the method of patent document 2 has room for improvement in the inactivation of the remaining formyl group.
The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide an immobilization method that suppresses the amount of ligand leakage and is excellent in deactivation of the remaining formyl group.
Means for solving the problems
The present inventors have intensively studied to solve the above problems, and as a result, they have found that a ligand can be more reliably immobilized on an insoluble substrate and the number of remaining formyl groups can be reduced by using 2 or more reducing agents, thereby completing the present invention.
That is, the present invention relates to the following [1] to [9 ].
[1] A method for immobilizing a ligand having an amino group to an insoluble substrate having a formyl group, the method comprising:
forming an imine by mixing the ligand and the insoluble substrate containing a formyl group; and
and a step of reducing the imine by using 2 or more reducing agents.
[2] The method according to the above [1], wherein the imine is reduced by adding the 2 or more reducing agents, respectively.
[3] The method according to the above [1] or [2], wherein the imine is reduced by using a borane complex having a Lewis base with a pKa of 6.5 or less as a ligand and then using another reducing agent as the reducing agent.
[4] The process according to the above [3], wherein the Lewis base having a pKa of 6.5 or less is a nitrogen-containing heterocyclic aromatic compound.
[5] The method according to any one of the above [1] to [4], wherein a peptide is used as the ligand.
[6] The method according to [5] above, wherein the peptide is capable of antibody-specific binding.
[7] The method according to any one of the above [1] to [6], wherein the insoluble substrate containing formyl groups is composed of at least 1 selected from the group consisting of polysaccharides, synthetic polymers and glass.
[8] The method according to any one of the above [1] to [7], wherein the shape of the formyl insoluble substrate is at least 1 selected from porous particles, monoliths, and porous membranes.
[9] A method for purifying a target compound, the method comprising:
a step of producing an adsorbent by immobilizing the ligand to the insoluble substrate containing a formyl group by the method according to any one of [1] to [8 ];
a step of bringing a liquid mixture containing the target compound into contact with the adsorbent to thereby adsorb the target compound to the adsorbent; and
separating the target compound adsorbed on the adsorbent from the adsorbent.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, it is possible to simultaneously achieve significant suppression of the amount of leakage of the ligand and sufficient inactivation of the remaining formyl group. Thus, the method of the present invention can produce a specific adsorbent capable of obtaining a high-purity target compound with a reduced amount of impurities mixed therein, and is industrially very excellent.
Detailed Description
One embodiment of the present invention will be described below, but the present invention is not limited thereto.
1. Introduction of formyl group into insoluble substrate
When an insoluble substrate containing formyl groups can be obtained commercially or the like, this step is not necessary, but when it cannot be obtained, formyl groups are introduced into the insoluble substrate according to a known method.
The insoluble base material is not particularly limited as long as it is insoluble in a solvent such as water that contains a mixed solution of the target compound and can adsorb the target compound. Examples thereof include: porous particles used for a chromatography packing material, biosensors used for analytical equipment for detecting a target compound, monoliths used for separation and recovery, analysis, and the like of a target compound, porous membranes used for separation and recovery of a target compound, removal of foreign substances, and the like, chips such as protein microarrays, and the like. Examples of the biosensor of the analytical device include a sensor chip of an analytical device using surface plasmon resonance or biofilm interference.
The material constituting the insoluble base material is not particularly limited as long as it is insoluble in a solvent such as a mixed solution containing a target compound, such as water, and examples thereof include: polysaccharides such as cellulose, agarose, dextran, starch, pullulan, chitosan (chitin), chitin (chitin), etc.; synthetic polymers such as poly (meth) acrylic acid, poly (meth) acrylate, polyacrylamide, and polyvinyl alcohol; silica glass, borosilicate glass, optical glass, soda-lime glass, and the like. In addition, the surface of a substrate made of a synthetic polymer having no functional group, such as polystyrene or a styrene-divinylbenzene copolymer, may be coated with a polymer material having a reactive functional group, such as a hydroxyl group. Examples of such a polymer material for coating include graft copolymers such as copolymers of monomers having hydroxyethyl methacrylate or polyoxyethylene chains and other polymerizable monomers having reactive functional groups. Among the above materials, polysaccharides, polyvinyl alcohol, and the like are preferably used because active groups can be easily introduced to the surface of the substrate.
As the shape of the insoluble base material, there can be mentioned: porous particles, monoliths, porous membranes.
The size of the porous particles as the insoluble substrate can be appropriately adjusted, and is preferably 20 μm or more and 1000 μm or less in volume average particle diameter, for example. When the volume average particle diameter is 20 μm or more, the back pressure (back pressure) at the time of packing in the column can be suppressed to be low. On the other hand, when the volume average particle diameter is 1000 μm or less, the surface area increases, and the amount of the target compound adsorbed increases. The volume average particle diameter is more preferably 30 μm or more, still more preferably 40 μm or more, yet more preferably 50 μm or more, yet more preferably 250 μm or less, yet more preferably 125 μm or less, yet more preferably 100 μm or less, and yet more preferably 85 μm or less. The volume average particle diameter of the porous particles can be determined by measuring the particle diameters of 100 porous particles selected at random. The particle size of each porous particle can be measured by taking a photomicrograph of each porous particle, storing the photomicrograph as electronic data, and using particle size measurement software (for example, "Image-Pro Plus" manufactured by Media Cybernetics). In order to improve strength and the like, the porous particles are preferably crosslinked by a polyfunctional compound by a usual method.
The monolith is a kind of porous continuous structure, and is a sponge-like structure in which a skeleton of a support structure and a hollow hole are integrated. The monolithic material has excellent mass transfer property and pressure flow rate characteristic, and can improve the adsorption efficiency and separation efficiency of a target compound, improve the liquid passing property or improve the detection sensitivity by controlling the size of a pore and the size of a framework. The structure of a porous substance having a continuous structure can be determined by confirming that different cross sections have pores having the same structure by observation with a scanning electron microscope or the like.
Examples of the porous membrane include porous membranes having a flat membrane, a hollow fiber, a deep filter structure, and the like.
In an insoluble substrate having pores such as monolith and porous membrane, the pore diameter can be appropriately adjusted depending on the target compound to be captured, the liquid passage speed, and the like, and may be, for example, 1nm or more and 10 μm or less. For example, when the target compound is an antibody or an antibody fragment, the pore diameter is particularly preferably 10nm or more and 300nm or less.
As a method for forming the raw material into an insoluble base material, a known method can be used. For example, in the case of porous particles, the porous particles can be formed by dispersing a solution or dispersion of the raw polymer in a fat or oil to form droplets, and then contacting the droplets with a solvent capable of mixing with the solvent of the solution or dispersion, such as alcohol or an alcohol-water mixture.
In order to introduce formyl groups into the insoluble substrate, functional groups of a raw material or a coating material constituting the insoluble substrate can be used. For example, when polysaccharides are used as a raw material, a large number of hydroxyl groups are present. An epoxy group can be introduced by reacting a halohydrin such as epichlorohydrin with the hydroxyl group. Alternatively, when a polyepoxide compound is used as a crosslinking agent, it is considered that unreacted epoxy groups remain. The epoxy group can be easily opened by an acidic aqueous solution or an alkaline aqueous solution. The ring-opened epoxy group is a 1, 2-diol group, and the 1, 2-diol group can be oxidized by an oxidizing agent to form a formyl group.
As the oxidizing agent for oxidizing the hydroxyl group to the formyl group, for example, periodic acid or periodate can be used. As periodate salts, mention may be made of: sodium periodate and potassium periodate.
The content of formyl groups in the insoluble base material containing formyl groups is not particularly limited, but is preferably 0.5. mu. mol or more and 100. mu. mol or less per 1mL of the insoluble base material containing formyl groups. When the content of formyl groups is 0.5. mu. mol or more per 1mL of an insoluble substrate containing formyl groups, the ligand can be immobilized efficiently, and when the substrate is used as an adsorbent, the amount of the target substance adsorbed increases, which is preferable. Although the reason is not clear, surprisingly, when the content of the formyl group is 100. mu. mol or less per 1mL of the insoluble substrate containing the formyl group, the amount of the target substance adsorbed is likely to increase. In addition, when a method of introducing a formyl group by reacting periodic acid and/or a periodate is used, it is preferable that the strength of the formyl group-containing insoluble base is easily increased when the formyl group content is 100. mu. mol or less per 1mL of the formyl group-containing insoluble base. The content of the formyl group is more preferably 1. mu. mol or more, still more preferably 1.5. mu. mol or more, yet more preferably 2. mu. mol or more, yet more preferably 75. mu. mol or less, yet more preferably 50. mu. mol or less, yet more preferably 40. mu. mol or less per 1mL of the insoluble substrate containing a formyl group. The content of the formyl group can be adjusted by, for example, the time and temperature of the reaction for introducing the formyl group, the concentration of a formylating agent such as periodic acid and/or periodate, and the like. In the present invention, the volume of the insoluble base material containing formyl groups as a reference for the content of formyl groups and the like is the volume of the entire structure including pores and a skeleton in the monolith, porous membrane and the like, and the porous particles and the like are the tap volumes when not particularly described. The tap volume is a volume in which a slurry containing porous particles and a dispersion medium such as water is dropped into a measuring vessel and is settled to a state where the volume is not reduced any more while applying vibration.
The formyl group content can be evaluated by adding a phenylhydrazine solution to an insoluble substrate containing formyl groups, stirring at 40 ℃ for 1 hour, measuring the absorption spectrum of the supernatant after the reaction with an ultraviolet-visible spectrophotometer, and measuring the amount of phenylhydrazine reduction from a phenylhydrazine calibration curve.
2. Step of introducing amino group into ligand
When the ligand has an amino group, the present step is not required, and when the ligand does not have an amino group, an amino group is introduced.
In the present invention, the ligand bound to the insoluble substrate refers to, for example, a substance capable of selectively binding to a target compound from a certain group of molecules based on the affinity between the molecules specific to the target compound. The ligand is a substance having affinity for the target compound, and examples thereof include: peptides, sugar chains, enzyme substrate compounds, DNA, and the like. In the present invention, a peptide refers to a compound in which 2 or more amino acids are bonded together by peptide bonds, and is a substance having specific affinity for a target compound, and examples thereof include: receptor proteins that bind to a substrate compound, antibodies to antigens, proteins having specific affinity for a target compound such as lectins that can bind to sugar chains, and antibody fragments such as subunits, domains, and Fab regions of proteins that retain specific affinity for a target compound.
Examples of the peptide that can be used as a ligand include an antibody affinity ligand. Examples of antibody affinity ligands include: protein a, protein G, protein L, protein H, protein D, protein Arp, protein Fc γ R, antibody binding synthetic ligands, and analogs thereof. In the present invention, the analogs of these antibody affinity ligands mean substances obtained by deleting, substituting and/or adding 1 or more amino acids constituting the above-mentioned protein a and the like, and are modifications retaining or improving the affinity for the target antibody or a fragment thereof, subunits and domains thereof retaining the affinity for the target antibody or a fragment thereof, as compared with the natural type. The upper limit of the number of mutations such as deletion in the above-described modified body depends on the amino acid and the like constituting the original peptide, and may be, for example, 20 or less, more preferably 10 or less or 5 or less. The number of mutations is preferably 1 or more.
When no amino group is present in the enzyme substrate compound or sugar chain, an amino group is introduced. It is easy for those skilled in the art to convert functional groups present in the substrate compound and the sugar chain into amino groups and introduce amino groups using the functional groups. When the peptide to be used as a ligand has only an amino group at the N-terminus or a side chain amino group is not sufficiently present, a basic amino acid such as lysine or a derivative thereof may be introduced or substituted into an arbitrary site by gene recombination techniques, synthetic techniques, or the like. In addition, in the case where there is no or insufficient available amino group in DNA or sugar, an amino group can be introduced by the same technique.
In the present invention, the compound targeting the ligand is a target for purification and detection, and the ligand is not particularly limited as long as it can specifically bind thereto. Examples thereof include: protein a, protein G, protein L, protein H, protein D, protein Arp, protein Fc γ R, immunoglobulin G (igg) bound to an antibody-binding synthetic ligand, and immunoglobulin G derivatives; a glycoprotein that binds to a lectin; plasminogen (plasminogen) bound to lysine (リシン); biotin bound to avidin; a protease bound to a protease inhibitor; a triazine-binding nucleotide binding protein; casein or src kinase bound to tyrosine, and the like. The immunoglobulin G derivative includes antibody fragments such as Fab.
3. Reaction procedure of ligand and insoluble substrate
In this step, an imine is formed by mixing a ligand having affinity specific to the target compound and an amino group with an insoluble substrate containing a formyl group. More specifically, the imino group is formed by reacting a formyl group of the insoluble substrate with an amino group of the ligand.
The pH of the reaction solution in the imidization reaction between the ligand and the insoluble substrate is preferably in the range of 7.0 or more and less than 13.0 in order to increase the immobilization amount and/or the immobilization rate of the amino group-containing ligand.
The solvent for the imidization reaction is preferably a buffer solution from the viewpoint of pH stability. The buffer solution usable in the present invention is not particularly limited, and a known buffer solution can be preferably used.
The temperature of the imidization reaction can be appropriately adjusted, and is preferably-10 ℃ or higher and 50 ℃ or lower. The reaction temperature is preferably-10 ℃ or higher from the viewpoint of the fluidity of the reaction solution, and if it is 50 ℃ or lower, the ligand and the formyl group of the insoluble substrate are less likely to be inactivated, so that it is preferable. The reaction temperature is more preferably-5 ℃ or higher, still more preferably 0 ℃ or higher, still more preferably 45 ℃ or lower, and still more preferably 40 ℃ or lower.
The reaction time may be sufficient for reacting the ligand with the insoluble substrate, and may be specifically determined by preliminary experiments or the like, and may be, for example, 1 hour or more and 50 hours or less.
After the reaction, the post-treatment may be carried out according to a usual method, and since the imino group is relatively unstable, it is preferable to proceed directly to the next step.
4. Reduction step of imino group
In this step, the imino group formed between the amino group of the ligand and the formyl group of the insoluble substrate in the previous step is reduced. In this step, by reacting with 2 or more reducing agents, the imino group formed by the formyl group of the insoluble substrate and the amino group of the ligand and the unreacted remaining formyl group can be sufficiently reduced, and the ligand can be more reliably immobilized on the insoluble substrate, and the remaining formyl group can be reduced, and it is presumed that the leakage of the ligand can be significantly suppressed and the risk of nonspecific adsorption by the remaining formyl group can be reduced. Further, since the effect can be exhibited with a small amount of reducing agent, the cost and the environmental load can be suppressed, and the method is industrially excellent.
As described above, by using 2 or more reducing agents, the amount of ligand leakage can be significantly reduced. More specifically, the leakage amount of the ligand can be set to 200ng/mL or less under the conditions of the examples described later. The leakage amount is more preferably 150ng/mL or less, and still more preferably 100ng/mL or less.
In addition, if a formyl group remains on the insoluble substrate after ligand immobilization, a compound other than the target compound reacts with or adsorbs nonspecifically to the formyl group, and there is a possibility that only the target compound cannot be selectively adsorbed. The remaining amount of formyl groups is preferably 8. mu. mol or less, more preferably 5. mu. mol or less, and still more preferably 3. mu. mol or less per 1mL of the insoluble substrate.
The present inventors have found through experiments that not only can the amount of ligand leakage be reduced, but also the amount of remaining formyl groups can be further reduced by using 2 or more reducing agents in the reduction step of an imino group. Surprisingly, it was found through experiments that the above-mentioned effect is further improved by adding each reducing agent separately in sequence, as compared to combining the above-mentioned 2 or more reducing agents and using them simultaneously.
The reducing agent usable in the present invention is not particularly limited, and for example, a borane complex can be used. As more specific examples, there may be mentioned: 4- (dimethylamino) pyridine borane, N-ethyldiisopropylamine borane, N-ethylmorpholine borane, N-methylmorpholine borane, N-phenylmorpholine borane, lutidine borane, triethylamine borane, or trimethylamine borane, 4- (dimethylamine) pyridine borane, N-ethyldiisopropylamine borane, N-ethylmorpholine borane, N-methylmorpholine borane, N-phenylmorpholine borane, lutidine borane, ammonia borane, dimethylamine borane, pyridine borane, 2-picoline borane (. alpha. -picoline borane), 3-picoline borane (. beta. -picoline borane), 4-picoline borane (. gamma. -picoline borane), N 'N-diethylaniline borane, N' N-diisopropylethylamine borane, N-ethylmorpholine borane, N-dimethylmorpholine borane, N-dimethyldiisopropylamine borane, N-ethylborane, N-dimethylborane, 2, 6-dimethylpyridine borane, borane amine, trisdimethylaminoborane, trimethylaminoborane, borazine, 1,3, 5-trimethylborazine, 2,4, 6-trimethylborazine, hexamethylborazine, sodium cyanoborohydride, sodium triacetoxyborohydride, and the like.
Further, among the borane complex reducing agents, the borane complex reducing agent having a lewis base with a pKa of 6.5 or less as a ligand can be used to effectively reduce the amount of ligand leakage. The pKa of the lewis base as the borane complex having the lewis base as a ligand is preferably 6.4 or less, more preferably 6.3 or less, and further preferably 6.2 or less. On the other hand, the lower limit of the pKa is not particularly limited, but it is considered that the amount of ligand leakage of the adsorbent tends to decrease as the borane complex having a lewis base with a lower pKa is used, but when the pKa is too low, there is a risk that the complex is hardly formed with borane, and it is preferably 0.2 or more, more preferably 0.5 or more or 1.0 or more, further preferably 2.0 or more, 3.0 or more, 4.0 or more, and further preferably 5.0 or more.
The lewis base having a pKa of 6.5 or less used in the present invention is a compound capable of supplying an electron pair to borane to form a complex, and is a compound that exerts a reducing action. Examples thereof include: amines, phosphines, phenols, amides, ureas, oximes.
When an unshared electron pair of a nitrogen atom is conjugated to an aromatic ring, pKa tends to decrease. Therefore, as the lewis base having a pKa of 6.5 or less used in the present invention, there can be mentioned: a nitrogen-containing heterocyclic aromatic compound, and/or an aromatic hydrocarbon compound having an amino group as a substituent.
The "nitrogen-containing heterocyclic aromatic compound" in the present invention is an aromatic compound containing at least 1 nitrogen atom in the aromatic ring, and refers to a compound having a pKa value of 6.5 or less, and examples thereof include: 5-membered nitrogen-containing heterocyclic aromatic compounds such as pyrrole; 6-membered nitrogen-containing heterocyclic aromatic compounds such as pyridine, pyridazine, pyrimidine and pyrazine; and condensed nitrogen-containing heterocyclic aromatic compounds such as quinoline, isoquinoline, phthalazine, quinazoline, and quinoxaline.
The "aromatic hydrocarbon compound having an amino group as a substituent" is an aromatic ring in which 1 or more amino groups as substituents are directly bonded to an aromatic ringThe hydrocarbon compound has a pKa value of 6.5 or less. Examples of the amino group include: -NH2Mono (C)1-6Alkyl) amino, di (C)1-6Alkyl) amino. The number of amino groups as a substituent tends to be larger as the number of substitution increases, and thus the pKa value tends to be larger, and is preferably 1 or 2. As the aromatic hydrocarbon compound, for example: c for benzene, naphthalene, biphenyl, etc6-12An aromatic hydrocarbon compound.
The nitrogen-containing heterocyclic aromatic compound may optionally have a substituent containing an amino group as long as the pKa value is 6.5 or less, and the aromatic hydrocarbon compound may optionally have a substituent other than an amino group as long as the pKa value is 6.5 or less. As the substituent other than amino group, there may be mentioned those selected from C1-6Alkyl radical, C1-61 or more of alkoxy, hydroxyl, halogeno, cyano and nitro.
In fact, since the pKa value varies depending on the type and number of the substituent, a lewis base having a pKa value of 6.5 or less can be selected based on the data on which the pKa value is recorded, actual measurement, and the like. For example, the nitrogen-containing heterocyclic aromatic compound and aromatic hydrocarbon compound include: pyridine; picolines such as α -picoline, β -picoline, and γ -picoline; diphenylamine; toluidines such as o-toluidine, m-toluidine, and p-toluidine; pyrrole, but not limited thereto.
Further, the aliphatic amine may have a pKa value of 6.5 or less depending on the kind and the number of the substituent. For example, aliphatic amines having pKa values of 6.5 or less include: hydroxylamines or alkoxyamines such as hydroxylamine, methoxyamine, N-methylhydroxylamine, and N, O-dimethylhydroxylamine; cyanoC such as cyanomethyldiethylamine, bis (cyanomethyl) amine, bis (cyanoethyl) amine1-6An alkyl amine.
Examples of the phosphine having a pKa value of 6.5 or less include tertiary phosphine, secondary phosphine, and primary phosphine having an electron-withdrawing group. As the tertiary phosphine having an electron-withdrawing group, for example: 2-cyanoethyl-di (C)1-6Alkyl) phosphines, phenyl-bis (C)1-6Alkyl) phosphines, bis (2-cyanoethyl) C1-6Alkyl phosphine, triphenyl phosphine, and tris (2-cyanoethyl)) A phosphine. Examples of secondary phosphines include: two (C)1-6Alkyl) phosphines, diphenylphosphine and bis (2-cyanoethyl) phosphine. Examples of the primary phosphine include C1-6An alkyl phosphine.
Examples of the phenol having a pKa value of 6.5 or less include phenols having an electron-withdrawing substituent at the ortho-position or para-position. For example, 2, 4-dinitrophenol, 2-chlorophenol, 2-bromophenol, 4-nitrophenol can be used.
Examples of amides having a pKa value of 6.5 or less include: cyanamide, C1-6Alkyl cyanamide, acetamide.
Examples of the urea having a pKa value of 6.5 or less include: urea, nitrourea and thiourea.
Examples of the oxime having a pKa value of 6.5 or less include: oxamido oximes, benzamide oximes, alpha-phenyl acetamide oximes, succinamide oximes, and toluamide oximes.
The borane complex can be usually produced by reacting diborane produced from sodium borohydride with a lewis base as a ligand.
The mode of adding 2 or more reducing agents to the reaction solution containing imine is not particularly limited, and 2 or more reducing agents may be added simultaneously, and more preferably, they are added sequentially. When the reducing agents are added in order, the order of addition is not particularly limited and can be used, and for example, the reducing agent used first includes a borane complex reducing agent having a lewis base having a pKa of 6.5 or less as a ligand. As the borane complex reducing agent having a Lewis base having a pKa of 6.5 or less as a ligand, the borane complex reducing agents exemplified above can be used, and examples thereof include pyridine borane and 2-methylpyridine borane. The other reducing agent may be used without particular limitation, and examples thereof include: dimethylamine borane, sodium triacetoxyborohydride, sodium cyanoborohydride. The number of reducing agents to be used is preferably 5 or less, preferably 4 or less, or 3 or less, and may be 2.
In the case where 2 or more reducing agents are added in sequence, the other reducing agents may be added immediately after 1 reducing agent is added, and preferably, the other reducing agents are added at intervals after 1 reducing agent is added. The time interval may be, for example, 10 minutes or more and 24 hours or less. The reaction solution may be allowed to stand, preferably stirred, between the addition of 2 or more reducing agents.
The solvent for the reduction reaction is preferably an aqueous solvent. Examples of the aqueous solvent include: water; aqueous solutions such as buffers; a water-miscible organic solvent; or a mixed solvent of an aqueous solution and a water-miscible organic solvent. The water-miscible organic solvent is an organic solvent capable of being mixed with water indefinitely, and examples thereof include: lower alcohol solvents such as methanol, ethanol, and isopropanol; amide solvents such as dimethylformamide and dimethylacetamide; sulfoxide solvents such as dimethyl sulfoxide.
In the reduction reaction in this step, when an aqueous solvent is used, denaturation and alteration of the immobilized ligand can be suppressed as compared with the case of using an organic solvent, and therefore, this is preferable. Among them, since the amine-borane complex is insoluble in water, an appropriate amount of a water-miscible organic solvent can be added depending on the water solubility of the amine-borane complex to be used. The concentration of the water-miscible organic solvent in the aqueous solvent is, for example, preferably 70% by mass or less, and more preferably 50% by mass or less. The concentration is preferably 2% by mass or more, and more preferably 5% by mass or more, for dissolving the amine-borane complex.
The pH of the reaction solution in the reduction reaction in this step is preferably in the range of 2 or more and less than 12. When the pH is 2 or more, the decomposition of the imino group and the inactivation of the borane complex by the reaction with water can be more reliably suppressed. On the other hand, when the pH is less than 12, the reaction of the borane complex can be further promoted. The pH is more preferably 3 or more, still more preferably less than 10, and still more preferably less than 9.
The reaction temperature, the reaction time, and the like may be conditions under which the reduction can be sufficiently performed, and may be determined by preliminary experiments and the like, and may be, for example, 1 hour or more and 50 hours or less, or 0 ℃ or more and 50 ℃ or less.
After the reaction, it is preferable to remove the reagent other than the ligand covalently immobilized on the insoluble substrate by the method of the present invention by washing the adsorbent. The cleaning agent and the cleaning method are not particularly limited, but it is preferable to add or stir at least 1 kind of solution containing water, acetic acid, alcohol, various organic solvents, an aqueous solution having a pH of 2 to 13, sodium chloride, potassium chloride, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, a buffer, a surfactant, urea, guanidine hydrochloride, other regenerants, and the like. In addition, when the same or different solution is used for a plurality of times of cleaning, the leakage amount of the ligand is further reduced, which is preferable.
The ligand-immobilized substrate of the present invention preferably has a ligand-introduced amount of 1mg to 500mg per 1mL of an insoluble substrate containing a formyl group. The amount of ligand introduced is preferably 500mg or less because the amount of the ligand adsorbed to the target compound increases when 1mg or more of the insoluble substrate containing a formyl group is 1mL, and the production cost can be suppressed. The ligand-introducing amount is more preferably 2mg or more, further preferably 3mg or more, further preferably 4mg or more, further preferably 120mg or less, further preferably 60mg or less, and further preferably 30mg or less per 1mL of the insoluble base material containing a formyl group.
The amount of ligand introduced into the ligand-immobilized substrate of the present invention is preferably 0.01. mu. mol to 15. mu. mol per 1mL of the insoluble substrate containing a formyl group. When the amount of the ligand to be introduced is 0.01. mu. mol or more per 1mL of the insoluble substrate containing a formyl group, the amount of the ligand adsorbed to the target compound increases, and therefore, 15. mu. mol or less is preferable because the production cost can be suppressed. The ligand introduction amount is more preferably 0.03. mu. mol or more, further preferably 0.05. mu. mol or more, further preferably 0.1. mu. mol or more, further preferably 5. mu. mol or less, further preferably 2. mu. mol or less, further preferably 1. mu. mol or less per 1mL of the insoluble base material containing a formyl group.
The amount of ligand introduced can be determined by: the absorbance of the ligand-derived ligand in the supernatant of the reaction solution before and after the immobilization reaction was measured, and the amount of unreacted ligand was determined from the difference between the measured values, assuming that all the other ligands were bound to the insoluble substrate. The amount of ligand introduced can also be determined by elemental analysis. For example, in the case of an amino group-containing ligand, the amount of the ligand introduced can be measured by analyzing the N content of the ligand-immobilized substrate.
5. Examples of use of the adsorbent
Since the adsorbent produced by firmly fixing the ligand to the insoluble substrate by the method of the present invention described above significantly suppresses leakage of the ligand, when the adsorbent is used for purification of a target compound, contamination of the ligand into the target compound is significantly suppressed. In addition, since the remaining formyl groups are sufficiently inactivated, it is expected that the non-specific adsorption is small when the adsorbent is used for purification of a target compound.
In order to purify a target compound by using the adsorbent of the present invention, the target compound is selectively adsorbed on the adsorbent by bringing a liquid mixture containing the target compound into contact with the adsorbent. The method of contacting is not particularly limited, and for example, only the adsorbent may be added to the mixed solution and mixed, but a method of introducing the mixed solution into a column after packing the adsorbent in the column is efficient and convenient.
For example, a column having a diameter of 0.1cm to 2000cm and a height of 1cm to 5000cm is preferably used. When the diameter is 0.1cm or more and the height is 1cm or more, the target compound can be efficiently adsorbed. From the viewpoint of the accuracy and efficiency of adsorption, the diameter is preferably 2000cm or less, and the height is preferably 5000cm or less.
The contact time (tolerance time) between the liquid mixture containing the target compound and the adsorbent is preferably 1 minute or more from the viewpoints of the accuracy of adsorption and the durability of the apparatus. On the other hand, from the viewpoint of efficiency, the contact time is preferably 12 minutes or less. The contact time is more preferably 2 minutes or more, further preferably 3 minutes or more, further preferably 10 minutes or less, further preferably 9 minutes or less.
As for the specific adsorption conditions, for example, it is preferable to adjust the amount of the target compound adsorbed per 1mL of the adsorbent to 1mg or more. When the adsorption amount is 1mg or more, the purification can be efficiently performed. On the other hand, when the adsorption amount is 200mg or less, the target compound adsorbed thereon is easily eluted from the adsorbent. The adsorption amount is more preferably 10mg to 150mg, still more preferably 20mg to 130mg, and still more preferably 30mg to 100 mg.
The amount of the target compound adsorbed is not particularly limited, and may be determined as a static adsorption amount or a dynamic adsorption amount. For example, in the case of measuring the static adsorption amount, it can be determined by: 0.5mL of the adsorbent which had been sufficiently washed with a phosphate buffer solution of pH7.4 was brought into contact with a solution prepared by dissolving 70mg of the objective compound in 35mL of the same phosphate buffer solution of pH7.4, and the mixture was stirred at 25 ℃ for 2 hours, and then the amount of the objective compound in the supernatant was measured.
After the target compound is adsorbed on the adsorbent of the present invention, the adsorbent is preferably washed to remove the non-specific adsorbed substance. The cleaning conditions are not particularly limited, and it is preferable to sufficiently clean the target compound by using a buffer solution having a pH of about 6.0 to 8.0 or less, ultrapure water, pure water, reverse osmosis water, distilled water, or the like so as not to desorb the target compound.
Next, the target compound adsorbed on the adsorbent is separated, whereby a purified target compound can be obtained. In order to separate the target compound from the adsorbent, the adsorbent may be washed with a buffer solution having a pH of about 3.0 to 5.0, for example.
The application claims priority based on the Japanese patent application No. 2018-3100 filed on 12.1.2018. The specification of japanese laid-open application No. 2018-3100, filed on 12.1.2018, is incorporated herein by reference in its entirety.
Examples
The present invention will be described more specifically below with reference to examples, but the present invention is not limited to the following examples, and can be carried out by appropriately changing the examples within a range that can meet the gist described before and after, and all of them are included in the technical scope of the present invention.
Example 1: manufacture of adsorbent
Crosslinked cellulose particles (gel obtained by the method described in jp 2009-a No. 242770) were used as an insoluble substrate. 70mL of the insoluble substrate was thoroughly washed with 0.01M citric acid buffer solution (prepared by using trisodium citrate dihydrate manufactured by SATUMA KAKO, citric acid monohydrate manufactured by SATUMA KAKO, and reverse osmosis water) having a pH of 3.4 on a glass filter. Subsequently, the washed insoluble substrate was introduced into a centrifuge tube, and the same citric acid buffer was added thereto so that the total volume was 108 mL. To this, an aqueous solution obtained by dissolving 0.45g of sodium periodate (manufactured by Kishida Chemical Co.) in 17.6mL of reverse osmosis water was added, and stirred at 6 ℃ for 40 minutes, thereby oxidizing the 1, 2-diol group of the insoluble substrate into a formyl group. The formyl group-containing carrier was obtained by filtration using a glass filter and washing with a sufficient amount of reverse osmosis water.
The obtained formyl group-containing carrier was washed well with 15mL of a 0.9M aqueous solution of dipotassium phosphate (prepared using dipotassium phosphate manufactured by Miyasan chemical Co., Ltd. and reverse osmosis water) on a glass filter. Next, the washed formyl group-containing vector was introduced into a separable flask, and the same aqueous solution of dipotassium phosphate was added thereto so that the total liquid volume became 19 mL. Further, with reference to WO2011/118699, protein A having the amino acid sequence of SEQ ID NO. 2 described in the International publication was prepared. After 2.6mL of a 58g/L aqueous solution of the protein A was added to the formyl group-containing carrier, the pH was adjusted to 11 with a 2M aqueous sodium hydroxide solution (prepared using a 24% aqueous sodium hydroxide solution manufactured by Kyowa K.K.) and then stirred at 7 ℃ for 15 hours. Then, the supernatant was removed to give a total liquid volume of 19mL, and then a solution prepared by dissolving 48mg (0.45mmol) of α -picoline borane (manufactured by Wako pure chemical industries, Ltd.) in 3.2mL of ethanol (manufactured by Wako pure chemical industries, Ltd.) and an aqueous solution prepared by dissolving 0.24g (4.05mmol) of dimethylamine borane (manufactured by Mitsui science, Ltd.) in 2.2mL of reverse immersion water were added simultaneously. Subsequently, 2.4M aqueous citric acid (prepared using citric acid 1 hydrate and reverse osmosis water) was added to adjust the pH of the mixture to 7.6, and then the mixture was heated to 25 ℃ and stirred for 4 hours.
The obtained carrier was washed with reverse osmosis water on a glass filter, and washed with a 0.1M aqueous citric acid solution, a 0.05M aqueous sodium hydroxide +0.5M aqueous sodium sulfate mixture (sodium sulfate manufactured by shiitake chemical industries co., ltd.) and a citric acid buffer solution (0.5M trisodium citrate 2 hydrate + citric acid 1 hydrate, pH 6) in this order. Finally, the column was washed with reverse osmosis water until the conductivity of the washing rate solution became 5. mu.S/cm or less, to obtain a ligand-immobilized adsorbent.
Comparative example 1: manufacture of adsorbent
A ligand-immobilized adsorbent was obtained in the same manner as in example 1, except that dimethylamine borane was not used, and only a 10-fold amount of α -picoline borane solution was used.
Comparative example 2: manufacture of adsorbent
A ligand-immobilized adsorbent was obtained in the same manner as in example 1, except that only 1.1 times the amount of dimethylamine borane aqueous solution was used instead of α -picoline borane.
Example 2: manufacture of adsorbent
A ligand-immobilized adsorbent was obtained in the same manner as in example 1, except that after the addition of the solution of α -picoline borane, 2.4M aqueous citric acid was added to adjust the pH of the mixture to 7.6, the temperature was raised to 25 ℃, and the mixture was stirred for 1 hour, and then aqueous dimethylamine borane was added to the mixture and stirred for 3 hours.
Example 3: manufacture of adsorbent
A ligand-immobilized adsorbent was obtained in the same manner as in example 2, except that the α -picoline borane added first was changed to pyridine borane (0.45 mmol).
Example 4: manufacture of adsorbent
In example 1, a pyridine borane (0.45mmol) in ethanol (1.4mL) was added to a reaction solution (19mL) of protein A and a formyl group-containing carrier. Subsequently, 2.4M aqueous citric acid was added to adjust the pH of the reaction mixture to 7.6, and the temperature was raised to 25 ℃. After the reaction mixture was stirred for 1 hour, an aqueous solution of dimethylamine borane (3.6mmol) was added. After stirring for 3 hours, a solution of N' -N-diethylaniline borane (0.45mmol) in ethanol was further added and stirred for 1 hour.
The obtained carrier was washed with reverse osmosis water on a glass filter, and further washed with a 0.1M aqueous citric acid solution, a 0.05M aqueous sodium hydroxide +0.5M aqueous sodium sulfate mixture (sodium sulfate manufactured by shiitake chemical co., ltd.), and a citric acid buffer solution (0.5M trisodium citrate 2 hydrate + citric acid 1 hydrate, pH 6) in this order. Finally, the mixture was washed with reverse osmosis water until the conductivity of the washing filtrate became 5. mu.S/cm or less, to obtain a ligand-immobilized adsorbent.
Test example 1: determination of the amount of remaining formyl groups
And estimating the amount of formyl groups remained on the insoluble substrate according to the residual phenylhydrazine after the reaction by utilizing the reaction of the residual formyl groups and phenylhydrazine. Specifically, after washing 4mL of each adsorbent with 0.1M sodium phosphate buffer solution of pH8, the total amount was adjusted to 6mL, 2mL of 0.1M sodium phosphate buffer solution of pH8 containing phenylhydrazine dissolved therein was added, the mixture was stirred at 40 ℃ for 1 hour, the absorbance of the maximum absorption near 278nm of the reaction supernatant was measured by UV measurement, and the amount of phenylhydrazine consumed was estimated as the amount of the residual formyl group from the amount of phenylhydrazine in the supernatant obtained. The results are shown in Table 1.
Test example 2: determination of ligand leakage
The amount of ligand leakage was determined when human IgG was adsorbed to the ligand-immobilized adsorbents prepared in the above examples and comparative examples.
(1) Solutions of
The following liquids a to E and neutralized liquids were prepared and defoamed before use.
Solution A: PBS buffer pH7.4 prepared using Phosphate buffer (Phosphate buffered saline) (manufactured by Wako pure chemical industries, Ltd.) and reverse osmosis water
And B, liquid B: a35 mM aqueous solution of sodium acetate was adjusted to pH3.5 with acetic acid (both sodium acetate and acetic acid manufactured by Wako pure chemical industries, Ltd.)
And C, liquid C: 0.1M phosphoric acid aqueous solution prepared using phosphoric acid manufactured by Wako pure chemical industries and reverse osmosis water
And (3) liquid D: an IgG aqueous solution having a concentration of 3mg/mL prepared using a polyclonal antibody ("GAMMAGARD" manufactured by Baxter Co.) and the solution A
E, liquid E: 0.1M aqueous sodium hydroxide solution prepared with sodium hydroxide and reverse osmosis water
Neutralizing liquid: 2M aqueous Tris (hydroxymethyl) aminomethane solution prepared from Tris (hydroxymethyl) aminomethane (Sigma-Aldrich) and reverse osmosis water
(2) Filling and preparing
A column having a diameter of 0.5 cm. times.15 cm and packed with the adsorbent sample prepared in the above example or comparative example was connected to a column chromatography apparatus ("AKTAexplorer 100" manufactured by GE Healthcare). A15 mL-tube for collection containing 3mL of a neutralizing solution was placed in the fraction collector.
(3) IgG purification
15mL of the solution A was passed through the column, followed by 50mL of the solution D (aqueous IgG solution). Then, 21mL of the solution A was passed through the reaction vessel, and 12mL of the solution B was passed through the reaction vessel, whereby IgG was eluted. Then, the solution was passed through 9mL of solution C, 9mL of solution A, 15mL of solution E, and 15mL of solution A. The flow rate of the solution D was set to 0.5 mL/min, and the flow rate of the solution A, B, C, E was set to 1 mL/min, so that the contact time with the adsorbent was 6 minutes or 3 minutes.
(4) Determination of the leakage of ligand
To evaluate the amount of leakage of ligand, the amount of ligand contained in the IgG eluate was determined. Specifically, the eluate obtained in the above test example 2(3) was collected, the amount of IgG and the amount of ligand in the eluate were measured, and the ligand concentration leaked from the purified IgG was determined as the leakage amount. Ligand concentrations were determined by ELISA. The relationship between the type and method of addition of the reducing agent, the amount of leakage of the ligand and the amount of the residual formyl group is shown in Table 1.
Figure BDA0002576676960000171
The results shown in table 1 demonstrate that there is still room for improvement in the amount of ligand leakage or the amount of the remaining formyl group when only 1 reducing agent is used, but by using 2 or more reducing agents, the amount of the remaining formyl group is sufficiently reduced and the amount of ligand leakage can be suppressed. It was also confirmed that the effect was more excellent by using 2 or more reducing agents in sequence, respectively, without using them at the same time.

Claims (9)

1. A method for immobilizing a ligand having an amino group to an insoluble substrate having a formyl group, the method comprising:
forming an imine by mixing the ligand and the insoluble substrate containing a formyl group; and
and a step of reducing the imine by using 2 or more reducing agents.
2. The method of claim 1, wherein,
the imine is reduced by adding the 2 or more reducing agents, respectively.
3. The method of claim 1 or 2,
as the reducing agent, a borane complex having a lewis base with a pKa of 6.5 or less as a ligand is used, and then another reducing agent is used to reduce the imine.
4. The method of claim 3, wherein,
the lewis base having a pKa of 6.5 or less is a nitrogen-containing heterocyclic aromatic compound.
5. The method according to any one of claims 1 to 4,
peptides were used as the ligands.
6. The method of claim 5, wherein,
the peptides are capable of antibody specific binding.
7. The method according to any one of claims 1 to 6,
the insoluble base material containing formyl groups is composed of at least 1 selected from polysaccharides, synthetic polymers and glass.
8. The method according to any one of claims 1 to 7,
the shape of the formyl insoluble substrate is at least 1 selected from the group consisting of porous particles, monoliths, and porous membranes.
9. A method for purifying a target compound, the method comprising:
a step of producing an adsorbent by immobilizing the ligand to the insoluble substrate containing a formyl group by the method according to any one of claims 1 to 8;
a step of bringing a liquid mixture containing the target compound into contact with the adsorbent to thereby adsorb the target compound to the adsorbent; and
separating the target compound adsorbed on the adsorbent from the adsorbent.
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CN102239001A (en) * 2008-12-03 2011-11-09 株式会社钟化 Formyl group-containing porous support, adsorbent using same, method for producing same, and method for producing the adsorbent
WO2017034024A1 (en) * 2015-08-27 2017-03-02 株式会社カネカ Ligand immobilization method

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US5276062A (en) * 1989-09-29 1994-01-04 Rohm And Haas Company HPLC avidin monomer affinity resin
CN101513606A (en) * 2004-05-24 2009-08-26 株式会社资生堂 Affinity particle and affinity separation method
CN102239001A (en) * 2008-12-03 2011-11-09 株式会社钟化 Formyl group-containing porous support, adsorbent using same, method for producing same, and method for producing the adsorbent
WO2017034024A1 (en) * 2015-08-27 2017-03-02 株式会社カネカ Ligand immobilization method

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